Mosquito-borne pathogens cause enormous human suffering. The genome sequences of vector mosquitoes are important resources for understanding and exploitation of vector-specific processes to reduce the burden of disease. However, a major limitation to studies of these processes is the inadequacy of available genetic tools for manipulating gene expression in vivo. Our preliminary studies demonstrate the feasibility of a new method for DNA delivery to embryos based on key elements of female mosquito biology. Our studies document the persistence of plasmid DNA and reporter gene activity from the egg to the adult stage in both Aedes aegypti and Anopheles gambiae, following delivery of the plasmids to the staged females. We now propose to build on this background and test potential applications of the vertical delivery method. In Aim 1, we will optimize the method and determine whether transient expression of reporter genes can be used to identify adult female-specific promoters for the ovary and midgut. In Aim 2, we will determine whether vertical delivery of transposable element constructs can be used to efficiently generate transgenic lines of Ae. aegypti and An. gambiae. Successful completion of these aims will provide new methods that will greatly facilitate future analyses of mosquito biology. PUBLIC HEALTH RELEVANCE: Relevance to public health strategies for controlling mosquitoes and reducing the burden of vector borne disease include the release of transgenic mosquitoes that carry dominant lethal genes or anti- pathogen mechanisms. Important directions for the field also include the dissection of the olfactory pathways, the mechanisms of insecticide resistance, and the evolution and ecology of key behaviors associated with human biting. Our proposed study will have impacts on all of these areas and any others that benefit from the ability to analyze gene function within mosquitoes. Highly efficient generation of transgenic mosquitoes and new methods for transient analysis of gene expression will have significant and lasting impacts on both basic biological questions as well as applied approaches to disease control. .